A simple method to produceSynechocystisPCC6803 biofilm under laboratory conditions for electron microscopic and functional studies

Cyanobacteria can form biofilms in nature, which have ecological roles and high potential for practical applications. In order to study them we need biofilm models that contain healthy cells and can withstand physical manipulations needed for structural studies. At present, combined studies on the structural and physiological features of axenic cyanobacterial biofilms are limited, mostly due to the shortage of suitable model systems. Here, we present a simple method to establish biofilms using the cyanobacteriumSynechocystisPCC6803 under standard laboratory conditions to be directly used for photosynthetic activity measurements and scanning electron microscopy (SEM). We found that glass microfiber filters (GMF) with somewhat coarse surface features provided a suitable skeleton to formSynechocystisPCC6803 biofilms. Being very fragile, untreated GMFs were unable to withstand the processing steps needed for SEM. Therefore, we used polyhydroxybutyrate coating to stabilize the filters. We found that up to five coats resulted in GMF stabilization and made possible to obtain high resolution SEM images of the structure of the surface-attached cells and the extensive exopolysaccharide and pili network, which are essential features of biofilm formation. By using pulse-amplitude modulated variable chlorophyll fluorescence imaging, it was also demonstrated that the biofilms contain photosynthetically active cells. Therefore, theSynechocystisPCC6803 biofilms formed on coated GMFs can be used for both structural and functional investigations. The model presented here is easy to replicate and has a potential for high-throughput studies

Timely removal of exogenous cytokinin and the prevention of auxin transport from the shoot to the root affect the regeneration potential of Arabidopsis roots

In vitro regeneration of Arabidopsis from roots is generally achieved via indirect organogenesis. First, transdifferentiation of lateral root primordia to calli is achieved by a balanced auxin-to-cytokinin ratio that is followed by the induction of shoot meristem formation using a high cytokinin level. Here we demonstrate that if the root explants were transferred onto a hormone-free medium after a transient (4-days) cytokinin treatment, embryogenic marker genes (LEC1, LEC2, FUS3) started to be expressed. App. 50% of the regeneration foci developed into plantlets with trichome-less cotyledon-like leaves. Moreover, the somatic embryogenesis defective lec1 mutant could regenerate only shoots with trichome-bearing leaves under this condition. Based on these observations, the mixed accomplishment of shoot organogenesis and somatic embryogenesis is hypothesized in the Arabidopsis root explants cultured under hormone-free conditions following cytokinin induction. Using whole seedlings instead of root explants in the same experimental set up, no regenerates were formed on the roots. Applying the auxin transport inhibitor TIBA to the root-to-shoot junction of the seedlings, the regeneration ability of the root could be restored. The observations indicate that shoot-derived endogenous auxin blocks the cytokinin-induced regeneration process in the roots of whole seedlings. The expression of the wound-induced transcription factor WIND1 could be detected in the roots of unwounded seedlings if the shoot-to-root auxin transport was inhibited. Manipulating the exogenous cytokinin level together with the endogenous shoot-to-root auxin transport therefore could mimic the effect of wounding (removal of shoot) on plant regeneration from roots. Key message Transferring root explants from high cytokinin to hormone-free conditions resulted in the expression of embryogenic markers. Inhibiting the shoot-to-root auxin transport had similar effect on regeneration as wounding

A foszfatidilglicerol szerepe fotoszintetikus membránok szerkezetének kialakításában = The essential role of phosphatidylglycerol in the formation of photosynthetic membrane structure

A negatív töltéssel rendelkező lipidek közül a foszfatidilglicerol (PG) jelentős szerepet tölt be. 1. Létrehoztuk egy FG szintézisben gátolt Synechocytis PCC6803 mutánst melyben megnéztük a CP43 kapcsolódását a kettes fotokémiai rendszer reakció centrumához. Ezt a mutánst egy, a fénybegyűjtő rendszer nélküli mutánssal hoztuk létre, a cdsA gén inaktiválásával. Megállapítottuk, hogy az FG kiürülés hatására a CP43 fehérje bekötődése gátolt. Megállapítottuk, hogy az FG kiürülés csökkenti a cianobaktérium sejtek oxigénfejlesztő képességét. 2. Megállapítottuk, hogy az FG kiürülés megváltoztatja a fotoszintetikus membrán felszíni töltését. 3. Az FG molekulák hatására bekövetkező fotoszintetikus aktivitást a reakció centrumok megváltozása okozza. A reakció centrumok fényérzékennyé vállnak. Ez ellen a sejtek megnövekedett karotin tartalommal védekeznek. A myxoxantin és az echinenon mennyisége nő meg. 4. A Syenechococcus PCC PCC7942 cdsA mutáns létrehozásával igazoltuk, hogy az FG molekulák szerepe a fotoszintetikus szervezetekben általánosítható. Megállapítottuk, hogy az FG kiürülés a különböző törzseken különböző mértékben hat. 5 Az FG molekulák kiürülésének hatására bekövetkező stressz elleni protekció a karotinoid molekulák megjelenése fotoszintetikus reakció centrumokbanmellett megfigyeltük azt is, hogy az FG molekulák zsírsav összetétele megváltozik. A molekulák átalakulását tömegspektometriásan igazoltuk. Az eredményeinket egy áttekintő cikkünkben foglaltuk össze. | Among the negatively charged lipids phosphatidylglycerol (PG) plays an important role. We used transformable cyanobacterial strains.1n We used a Phycobilisome-less mutant of Synechocytis PCC6803 was transformed with inactivated cdsA and the synthesis of PG was blocked. Binding of CP43 to the reaction center complex was investigated. The oxygen evolving activity of mutant was reduced. 2. The PG-depletion affected the surface charge of membranes. 3. PG-depletion induced sensitivity of cells which was compensated by elevated carotenoid content, mainly myxoxanthophyll and echinenone. 4. We generated Syenechococcus PCC PCC7942 cdsA mutant with which we could generalize the importance of PG in photosynthetic organizms. However, we could observe slight differencein different mutant. 5. Westudied PG remodeling by mas spectrometry

Lipid Polymorphism of the Subchloroplast—Granum and Stroma Thylakoid Membrane–Particles. II. Structure and Functions

In Part I, by using P-31-NMR spectroscopy, we have shown that isolated granum and stroma thylakoid membranes (TMs), in addition to the bilayer, display two isotropic phases and an inverted hexagonal (H-II) phase; saturation transfer experiments and selective effects of lipase and thermal treatments have shown that these phases arise from distinct, yet interconnectable structural entities. To obtain information on the functional roles and origin of the different lipid phases, here we performed spectroscopic measurements and inspected the ultrastructure of these TM fragments. Circular dichroism, 77 K fluorescence emission spectroscopy, and variable chlorophyll-a fluorescence measurements revealed only minor lipase- or thermally induced changes in the photosynthetic machinery. Electrochromic absorbance transients showed that the TM fragments were re-sealed, and the vesicles largely retained their impermeabilities after lipase treatments-in line with the low susceptibility of the bilayer against the same treatment, as reflected by our P-31-NMR spectroscopy. Signatures of H-II-phase could not be discerned with small-angle X-ray scattering-but traces of H-II structures, without long-range order, were found by freeze-fracture electron microscopy (FF-EM) and cryo-electron tomography (CET). EM and CET images also revealed the presence of small vesicles and fusion of membrane particles, which might account for one of the isotropic phases. Interaction of VDE (violaxanthin de-epoxidase, detected by Western blot technique in both membrane fragments) with TM lipids might account for the other isotropic phase. In general, non-bilayer lipids are proposed to play role in the self-assembly of the highly organized yet dynamic TM network in chloroplasts

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation

Carotenoids, versatile components of oxygenic photosynthesis.

Carotenoids (CARs) are a group of pigments that perform several important physiological functions in all kingdoms of living organisms. CARs serve as protective agents, which are essential structural components of photosynthetic complexes and membranes, and they play an important role in the light harvesting mechanism of photosynthesizing plants and cyanobacteria. The protection against reactive oxygen species, realized by quenching of singlet oxygen and the excited states of photosensitizing molecules, as well as by the scavenging of free radicals, is one of the main biological functions of CARs. X-ray crystallographic localization of CARs revealed that they are present at functionally and structurally important sites of both the PSI and PSII reaction centers. Characterization of a CAR-less cyanobacterial mutant revealed that while the absence of CARs prevents the formation of PSII complexes, it does not abolish the assembly and function of PSI. CAR molecules assist in the formation of protein subunits of the photosynthetic complexes by gluing together their protein components. In addition to their aforementioned indispensable functions, CARs have a substantial role in the formation and maintenance of proper cellular architecture, and potentially also in the protection of the translational machinery under stress conditions

The Interplay of Apoes with Syndecans in Influencing Key Cellular Events of Amyloid Pathology

Apolipoprotein E (ApoE) isoforms exert intricate effects on cellular physiology beyond lipid transport and metabolism. ApoEs influence the onset of Alzheimer's disease (AD) in an isoform-dependent manner: ApoE4 increases AD risk, while ApoE2 decreases it. Previously we demonstrated that syndecans, a transmembrane proteoglycan family with increased expression in AD, trigger the aggregation and modulate the cellular uptake of amyloid beta (A beta). Utilizing our previously established syndecan-overexpressing cellular assays, we now explore how the interplay of ApoEs with syndecans contributes to key events, namely uptake and aggregation, in A beta pathology. The interaction of ApoEs with syndecans indicates isoform-specific characteristics arising beyond the frequently studied ApoE-heparan sulfate interactions. Syndecans, and among them the neuronal syndecan-3, increased the cellular uptake of ApoEs, especially ApoE2 and ApoE3, while ApoEs exerted opposing effects on syndecan-3-mediated A beta uptake and aggregation. ApoE2 increased the cellular internalization of monomeric A beta, hence preventing its extracellular aggregation, while ApoE4 decreased it, thus helping the buildup of extracellular plaques. The contrary effects of ApoE2 and ApoE4 remained once A beta aggregated: while ApoE2 reduced the uptake of A beta aggregates, ApoE4 facilitated it. Fibrillation studies also revealed ApoE4 ' s tendency to form fibrillar aggregates. Our results uncover yet unknown details of ApoE cellular biology and deepen our molecular understanding of the ApoE-dependent mechanism of A beta pathology

Label-free LC-MS/MS identification of phosphatidylglycerol-regulated proteins in Synechocystis sp. PCC6803.

We present a proteomics dataset combining SDS-PAGE prefractionation and data-dependent LC-MS/MS that enables the identification of phosphatidylglycerol-regulated proteins in the pgsA- mutant of Synechocystis sp. PCC6803, a cyanobacterium strain that grows with this indispensable phospholipid added exogenously. We searched the acquired raw data against a composite protein sequence database of Synechocystis using Mascot, and employed Progenesis LC-MS software for label-free quantification based on extracted peptide intensitiesto detect changes in protein abundances upon phospholipid withdrawal. Protein identifications were validated using rigorous criteria, and our analysis of the dataset revealed 80 phosphatidylglycerol-regulated proteins involved in various cellular processes including photosynthesis, respiration, metabolism, transport, transcription, and translation. The data have been deposited to the ProteomeXchange with identifier PXD000363. This article is protected by copyright. All rights reserved